Contractile properties of slow and fast skeletal muscles from protease activated receptor‐1 null mice

Introduction: Protease‐activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow‐twitch soleus muscles and fast‐twitch extensor digitorum longus (EDL) muscles from PAR‐1 null and littermate control mice. Methods: Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry. Results: Soleus muscles from PAR‐1 null mice exhibited longer contraction times, a leftward shift in the force–stimulation frequency relationship, and decreased fatiguability compared with controls. PAR‐1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR‐1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls. Conclusions: The absence of PAR‐1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991–998, 2014     

Upregulation of MHC class I in transgenic mice results in reduced force‐generating capacity in slow‐twitch muscle

Expression of major histocompatibility complex (MHC) class I in skeletal muscle fibers is an early and consistent finding in inflammatory myopathies. To test if MHC class I has a primary role in muscle impairment, we used transgenic mice with inducible overexpression of MHC class I in their skeletal muscle cells. Contractile function was studied in isolated extensor digitorum longus (EDL, fast‐twitch) and soleus (slow‐twitch) muscles. We found that EDL was smaller, whereas soleus muscle was slightly larger. Both muscles generated less absolute force in myopathic compared with control mice; however, when force was expressed per cross‐sectional area, only soleus muscle generated less force. Inflammation was markedly increased, but no changes were found in the activities of key mitochondrial and glycogenolytic enzymes in myopathic mice. The induction of MHC class I results in muscle atrophy and an intrinsic decrease in force‐generation capacity. These observations may have important implications for our understanding of the pathophysiological processes of muscle weakness seen in inflammatory myopathies. Muscle Nerve, 2008     

Aging impairs regulation of ryanodine receptors from extensor digitorum longus but not soleus muscles

ABSTRACT: Introduction: Because impaired excitation‐contraction coupling and reduced sarcoplasmic reticulum (SR) Ca²⁺ release may contribute to the age‐associated decline in skeletal muscle strength, we investigated the effect of aging on regulation of the skeletal muscle isoform of the ryanodine receptor (RyR1) by physiological channel ligands. Methods: [³H]Ryanodine binding to membranes from 8‐ and 26‐month‐old Fischer 344 extensor digitorum longus (EDL) and soleus muscles was used to investigate the effects of age on RyR1 modulation by Ca²⁺ and calmodulin (CaM). Results: Aging reduced maximal Ca²⁺‐stimulated binding to EDL membranes. In 0.3 μM Ca²⁺, age reduced binding and CaM increased binding to EDL membranes. In 300 μM Ca²⁺, CaM reduced binding, but the age effect was not significant. Aging did not affect Ca²⁺ or CaM regulation of soleus RyR1. Discussion: In aged fast‐twitch muscle, impaired RyR1 Ca²⁺ regulation may contribute to lower SR Ca²⁺ release and reduced muscle function. Muscle Nerve 57 : 1022–1025, 2018     

Effects of moderate‐ and high‐intensity chronic exercise on brain‐derived neurotrophic factor expression in fast and slow muscles

Introduction: Brain‐derived neurotrophic factor (BDNF) protein expression is sensitive to cellular activity. In the sedentary state, BDNF expression is affected by the muscle phenotype. Methods: Eighteen Wistar rats were divided into the following 3 groups: sedentary (S); moderate‐intensity training (MIT); and high‐intensity training (HIT). The training protocol lasted 8 weeks. Forty‐eight hours after training, total RNA and protein levels in the soleus and plantaris muscles were obtained. Results: In the plantaris, the BDNF protein level was lower in the HIT than in the S group (P < 0.05). A similar effect was found in the soleus (without significant difference). In the soleus, higher Bdnf mRNA levels were found in the HIT group (P < 0.001 vs. S and MIT groups). In the plantaris muscle, similar Bdnf mRNA levels were found in all groups. Conclusions: These results indicate that high‐intensity chronic exercise reduces BDNF protein level in fast muscles and increases Bdnf mRNA levels in slow muscles. Muscle Nerve 53: 446–451, 2016     

Heat‐Stress effects on the myosin heavy chain phenotype of rat soleus fibers during the early stages of regeneration

Introduction: We investigated heat‐stress effects on the adult myosin heavy chain (MyHC) profile of soleus muscle fibers at an early stage of regeneration. Methods: Regenerating fibers in adult rats were analyzed 2, 4, or 6 days after bupivacaine injection. Rats were heat stressed by immersion in water (42 ± 1°C) for 30 minutes 24 hours after bupivacaine injection and every other day thereafter. Results: No adult MyHC isoforms were observed after 2 days, whereas some fibers expressed only fast MyHC after 4 days. Heat stress increased fast and slow MyHC in regenerating fibers after 6 days. Regenerating fibers expressing only slow MyHC were observed only in heat‐stressed muscles. Bupivacaine injection increased the number of Pax7⁺ and MyoD⁺ satellite cells in regenerating fibers, more so in heat‐stressed rats. Conclusion: The results indicate that heat stress accelerates fast‐to‐slow MyHC phenotype conversion in regenerating fibers via activation of satellite cells. Muscle Nerve 52 : 1047–1056, 2015     

Progressive motor unit loss in the G93A mouse model of amyotrophic lateral sclerosis is unaffected by gender

We examined whether there are gender differences in the progressive loss of functional motor units in SOD1^G93A transgenic mice. Isometric muscle and motor unit twitch contractions were recorded in fast‐ and slow‐twitch muscles in response to stimulation of the sciatic nerve. Using a modified motor unit number estimation technique (ITS‐MUNE), we found that motor unit numbers declined rapidly from 40 to 90 days of age during the asymptomatic phase of ALS in fast‐ but not slow‐twitch hindlimb muscles of both male and female mice. There was a corresponding decline in twitch and tetanic contractile forces of the fast‐twitch muscles. Gender did not affect the progressive loss of motor units and associated decline in force production. We conclude that gender does not alter progressive, muscle‐specific motor unit loss in ALS, even though gender does influence disease onset. Muscle Nerve 39: 318–327, 2009     

Twitch and M‐wave potentiation induced by intermittent maximal voluntary quadriceps contractions: Differences between direct quadriceps and femoral nerve stimulation

Introduction: The aim of this study was to investigate differences in twitch and M‐wave potentiation in the quadriceps femoris when electrical stimulation is applied over the quadriceps muscle belly versus the femoral nerve trunk. Methods: M‐waves and mechanical twitches were evoked using direct quadriceps muscle and femoral nerve stimulation between 48 successive isometric maximal voluntary contractions (MVC) from 10 young, healthy subjects. Potentiation was investigated by analyzing the changes in M‐wave amplitude recorded from the vastus medialis (VM) and vastus lateralis (VL) muscles and in quadriceps peak twitch force. Results: Potentiation of twitch, VM M‐wave, and VL M‐wave were greater for femoral nerve than for direct quadriceps stimulation (P < 0.05). Despite a 50% decrease in MVC force, the amplitude of the M‐waves increased significantly during exercise. Conclusions: In addition to enhanced electrogenic Na⁺‐K⁺ pumping, other factors (such as synchronization in activation of muscle fibers and muscle architectural properties) may significantly influence the magnitude of M‐wave enlargement. Muscle Nerve 48 : 920–929, 2013     

Gender‐specific neuromuscular adaptations to unloading in isolated rat soleus muscles

Introduction: The potential of gender to affect unloading‐induced neuromuscular adaptations was investigated. Methods: Twenty male and 20 female rats were assigned to control (CTL), or unloaded (UL) conditions. After 2 weeks of unloading, soleus muscles were removed, and neuromuscular function was assessed during a train of alternating indirect (neural) and direct (muscle) stimuli. Results: In rested muscle, strength showed significant (P ≤ 0.05) main effects for gender (male > female) and treatment (CTL > UL). By the end of the testing protocol, when muscles showed fatigue, gender‐related and treatment‐related differences in strength had disappeared. Neuromuscular transmission efficiency and strength suffered a greater decline during the testing protocol in males than females. Unloaded male muscles displayed greater contractile velocity than female muscles both when rested and fatigued. Conclusions: Gender affected unloading‐induced neuromuscular adaptations. The greater strength of rested male muscles was due to greater muscle mass and neuromuscular transmission efficiency. Muscle Nerve 54 : 300–307, 2016     

Lack of caspase‐3 attenuates immobilization‐induced muscle atrophy and loss of tension generation along with mitigation of apoptosis and inflammation

Introduction: Immobilization by casting induces disuse muscle atrophy (DMA). Methods: Using wild type (WT) and caspase‐3 knockout (KO) mice, we evaluated the effect of caspase‐3 on muscle mass, apoptosis, and inflammation during DMA. Results: Caspase‐3 deficiency significantly attenuated muscle mass decrease [gastrocnemius: 28 ± 1% in KO vs. 41 ± 3% in WT; soleus: 47 ± 2% in KO vs. 56 ± 2% in WT; (P < 0.05)] and gastrocnemius twitch tension decrease (23 ± 4% in KO vs. 36 ± 3% in WT, P < 0.05) at day 14 in immobilized vs. contralateral hindlimb. Lack of caspase‐3 decreased immobilization‐induced increased apoptotic myonuclei (3.2‐fold) and macrophage infiltration (2.2‐fold) in soleus muscle and attenuated increased monocyte chemoattractant protein‐1 mRNA expression (2‐fold in KO vs. 18‐fold in WT) in gastrocnemius. Conclusions: Caspase‐3 plays a key role in DMA and associated decreased tension, presumably by acting on the apoptosis and inflammation pathways. Muscle Nerve 47: 711–721, 2013     

Soleus and lateral gastrocnemius H‐reflexes during standing with unstable footwear

Introduction: Unstable footwear has been shown to increase lower extremity muscle activity, but the reflex response to perturbations induced by this intervention is unknown. Methods: Twenty healthy subjects stood in stable and unstable footwear conditions (presented randomly) while H‐reflex amplitude and background muscle activity were measured in the soleus and lateral gastrocnemius (LG) muscles. Results: Wearing unstable footwear resulted in larger H‐reflexes (normalized to the maximal M‐wave) for the LG (+12%; P = 0.025), but not for the soleus (+4%; P > 0.05). Background activity of both muscles was significantly higher in the unstable condition. Conclusions: The H‐reflex facilitation observed with unstable footwear was unexpected, as challenging postural conditions usually result in reflex depression. Increased muscle activity, decreased presynaptic inhibition, and/or more forward postural position may have (over‐)compensated the expected reflex depression. Differences between LG and soleus H‐reflex modulation may be due to diverging motor unit recruitment thresholds. Muscle Nerve 51 :764–766, 2015     

Activation of fast-twitch fibers assessed with twitch potentiation

Introduction: The augmentation of twitch response following brief muscle activation, called twitch potentiation, has been shown to be much more pronounced in fast‐twitch than in slow‐twitch fibers. We thus explored the possibility of twitch potentiation as a noninvasive measure of fast‐twitch fiber activation, by studying its dependence on the intensity of preceding contraction. Methods: Twitch contraction of plantar flexor muscles was evoked with supramaximal stimulation of the posterior tibial nerve, before and immediately after 6‐s voluntary contractions at intensities of 10–100% of maximal voluntary contraction (MVC). Results: Except for low‐intensity contractions (< 30%MVC), voluntary contraction induced twitch potentiation, the magnitude of which increased with increasing contraction intensity (P < 0.001). The shortened contractile process was associated with the potentiation. Conclusions: These results are consistent with the concept of “hierarchical order of fiber activation”, suggesting that the magnitude of twitch potentiation reflects the activation of fast‐twitch fibers during a brief contraction. Muscle Nerve 46: 218–227, 2012     

Specific impulse patterns regulate acetylcholinesterase activity in skeletal muscles of rats and rabbits

In rats, acetylcholinesterase (AChE) activity in the fast muscles is several times higher than in the slow soleus muscle. The hypothesis that specific neural impulse patterns in fast or slow muscles are responsible for different AChE activities was tested by altering the neural activation pattern in the fast extensor digitorum longus (EDL) muscle by chronic low-frequency stimulation of its nerve. In addition, the soleus muscle was examined after hind limb immobilization, which changed its neural activation pattern from tonic to phasic. Myosin heavy-chain (MHC) isoforms were analyzed by gel electrophoresis. Activity of the molecular forms of AChE was determined by velocity sedimentation. Low-frequency stimulation of the rat EDL for 35 days shifted the profile of MHC II isoforms toward a slower MHCIIa isoform. Activity of the globular G₁ and G₄ molecular forms of AChE decreased by a factor of 4 and 10, respectively, and became comparable with those in the soleus muscle. After hind limb immobilization, the fast MHCIId isoform, which is not normally present, appeared in the soleus muscle. Activity of the globular G₁ form of AChE increased approximately three times and approached the levels in the fast EDL muscle. In the rabbit, on the contrary to the rat, activity of the globular forms of AChE in a fast muscle increased after low-frequency stimulation. The results demonstrate that specific neural activation patterns regulate AChE activity in muscles. Great differences, however, exist among different mammalian species in regard to muscle AChE regulation. J. Neurosci. Res. 47:49–57, 1997. © 1997 Wiley-Liss, Inc.     

Effect of hind-limb suspension on young and adult skeletal muscle. I. Normal mice

The purpose of this study was to determine the effect of hind-limb suspension (HS) on morphometric, histologic, and contractile characteristics of fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles in adult and immature mice. Hind-limb suspension for 2 weeks was used to produce atrophy in two groups of mice, ages 4 and 12 weeks, with nonsuspended animals serving as controls. Young HS mice exhibited marked decreases in SOL weight, length, cross-sectional area (CSA), twitch and tetanic tensions, and rates of tension development and relaxation, with increases in fatigue resistance. HS reduced the diameter of both type I and IIA fibers, increased the percentage of type I fibers, and decreased the percentage of type IIA fibers in both young and adult SOL. Muscle weight, length, CSA, IIA and IIB fiber areas, and maximum rate of tetanic tension development were decreased in EDL of young HS mice; fatigue resistance and EDL half-relaxation times were increased. For most parameters evaluated, slow twitch muscle was more affected than fast twitch. HS affected contractile characteristics less than morphometric or histologic parameters. Rates of tension development and relaxation were the contractile parameters most affected by HS, and the time parameters of contraction were least affected. For all measurements young mice were more affected than adult mice.     

Temperature dependence of contraction characteristics in developing rat muscles

Contractions of rat extensor digitorum longus (EDL, a fast muscle) and soleus (SOL, a slow muscle) muscles of different ages (1-4 weeks) were recorded in vitro with direct stimulation and at different temperatures (range 35-10 degrees C). Twitch tension in 4-week-old EDL muscle increased in cooling from 35 to 20 degrees C (cooling potentiation); the tension decreased in further cooling below 20 degrees C. This pattern of temperature dependence of twitch tension was seen in fast muscles of all ages (1-4 weeks). Twitch tension in 4-week-old SOL muscle decreased monotonically in cooling from 35 to 10 degrees C (cooling depression). This pattern of cooling depression was not clearly evident in younger SOL muscles. There was a marked hysteresis in the temperature dependence of twitch tension in the 1-week-old SOL muscles. Tetanic tension was depressed by low temperature in both EDL and SOL muscles at 1 week and at 4 weeks of age. Results show that the processes concerned with contractile activation are nearly fully developed in the fast muscle fibers at an early age (1 week), whereas they develop later in the slow muscle fibers.     

Effects of tail suspension on serum testosterone and molecular targets regulating muscle mass

Introduction: The contribution of reduced testosterone levels to tail suspension (TS)‐induced muscle atrophy remains equivocal. The molecular mechanism by which testosterone regulates muscle mass during TS has not been investigated. Methods: Effects of TS on serum testosterone levels, muscle mass, and expression of muscle atrophy‐ and hypertrophy‐inducing targets were measured in soleus (SOL) and extensor digitorum longus (EDL) muscles after testosterone administration during 1, 5, and 14 days of TS in male mice. Results: TS produced an increase followed by a transient drop in testosterone levels. Muscle atrophy was associated with downregulation of Igf1 and upregulation of Mstn, Redd1, Atrogin‐1, and MuRF1 mRNA with clear differences in Igf1, Mstn, and MAFbx/Atrogin‐1 gene expression between SOL and EDL. Testosterone supplementation did not affect muscle mass or protein expression levels during TS. Conclusions The known anabolic effects of testosterone are not sufficient to ameliorate loss of muscle mass during TS. Muscle Nerve 52 : 278–288, 2015     

Proteomic analysis of mouse soleus muscles affected by hindlimb unloading and reloading

Introduction: Disuse muscle atrophy, induced by prolonged space flight, bed rest, or denervation, is a common process with obvious changes in slow‐twitch soleus muscles. Methods: Proteomic analysis was performed on mouse soleus subjected to hindlimb unloading (HU) and hindlimb reloading (HR) to identify new dysregulated proteins. Results: Following HU, the mass and cross‐sectional area of muscle fibers decreased, but they recovered after HR. Proteomic analyses revealed 9 down‐regulated and 7 up‐regulated proteins in HU, and 2 down‐regulated and 5 up‐regulated proteins in HR. The dysregulated proteins were mainly involved in energy metabolism, protein degradation, and cytoskeleton stability. Among the dysregulated proteins were fatty acid binding protein 3, α‐B crystalline, and transthyretin. Conclusions: These results indicate that muscle atrophy induced by unloading is related to activation of proteolysis, metabolic alterations toward glycolysis, destruction of myofibrillar integrity, and dysregulation of heat shock proteins (HSPs). The dysregulated proteins may play a role in muscle atrophy and the recovery process. Muscle Nerve 52 : 803–811, 2015     

Effect of resistance training on neuromuscular junctions of young and aged muscles featuring different recruitment patterns

To examine the effects of aging on neuromuscular adaptations to resistance training (i.e., weight lifting), young (9 months of age) and aged (20 months of age) male rats either participated in a 7‐week ladder climbing protocol with additional weight attached to their tails or served as controls (n = 10/group). At the conclusion, rats were euthanized and hindlimb muscles were quickly removed and frozen for later analysis. Longitudinal sections of the soleus and plantaris muscles were collected, and pre‐ and postsynaptic features of neuromuscular junctions (NMJs) were visualized with immunofluorescence staining procedures. Cross‐sections of the same muscles were histochemically stained to determine myofiber profiles (fiber type and size). Statistical analysis was by two‐way ANOVA (main effects of age and treatment) with significance set at P ≤ 0.05. Results revealed that training‐induced remodeling of NMJs was evident only at the postsynaptic endplate region of soleus fast‐twitch myofibers. In contrast, aging was associated with pre‐ and postsynaptic remodeling in fast‐ and slow‐twitch myofibers of the plantaris. Although both the soleus and the plantaris muscles failed to display either training or aging‐related alterations in myofiber size, aged plantaris muscles exhibited an increased expression of type I (slow‐twitch) myofibers in conjunction with a reduced percentage of type II (fast‐twitch) myofibers, suggesting early stages of sarcopenia. These data demonstrate the high degree of specificity of synaptic modifications made in response to exercise and aging and that the sparsely recruited plantaris is more vulnerable to the effects of aging than the more frequently recruited soleus muscle. © 2014 Wiley Periodicals, Inc.     

Changes in number and distribution of orthogonal arrays during postnatal muscle development

Quantitative freeze-fracture electron microscopy was utilized to study the changes in number and distribution of orthogonal arrays (aggregates of 6-7 nm particles) of the sarcolemmas of the fast twitch extensor digitorum longus (EDL) and slow twitch soleus muscles during the first few weeks of postnatal development in the rat. In the adult rat, orthogonal arrays are present in high densities in the fast twitch type II fibers but only in low densities in slow twitch type I fibers. In this study, the changing histochemical profiles of fiber types in the EDL and soleus muscles were also determined for the first month of postnatal development and correlated with the changing number and distribution of orthogonal arrays during the same time frame. At day 3 postpartum, EDL and soleus fibers possessed few orthogonal arrays. The developing EDL fibers rapidly acquired additional orthogonal arrays until the approximate adult number and distribution were attained at postpartum day 25. In contrast, the slow twitch soleus fibers rapidly acquired orthogonal arrays and type IIA fibers until day 35 when both were in excess of adult values. Subsequently, the number of arrays and type IIA fibers declined to normal adult ranges. We suggest that the patterns of development of orthogonal arrays and fiber types are different in the EDL and soleus because the types of innervating motor units are different in the two muscles. The EDL is innervated almost entirely by fast motor units throughout early development and maturity. The soleus, however, is initially innervated by a more heterogeneous population of motor neurons. Thus, during the period of polyneuronal innervation which occurs normally during the first weeks of postnatal development, many individual soleus fibers may possess simultaneous innervation by axons from different motor neuron types. These dual influences may be responsible for the irregular pattern of development of orthogonal arrays and type IIA fibers in developing soleus fibers. Later, as the adult pattern of monosynaptic innervation is developed, expected adult values of orthogonal arrays and fiber types are attained.     

Post‐activation potentiation: The neural effects of post—activation depression

Introduction: Our knowledge of the neurophysiology of post‐activation potentiation (PAP) is limited. The purpose of this study was to examine the effect of PAP on twitch torque and H‐reflex amplitude after a 10‐s maximal voluntary contraction (MVC). Methods: PAP measurements were assessed with the plantarflexors in a relaxed state and during a tonic contraction at 10% MVC. Results: The H‐reflex/maximum M‐wave ratio (H/M) decreased significantly (P < 0.05) and returned to baseline levels after 1 min. The decrement in H/M was depressed when the plantarflexors were active at 10% MVC, and the depression was more obvious in the lateral gastrocnemius than in the soleus muscle. Conclusions: The inhibition induced immediately after contraction could be attributed to post‐activation depression. We conclude that PAP after a 10‐s MVC cannot be attributed to increased motor neuron excitability through the reflex pathway as assessed by the H‐reflex technique. Muscle Nerve 52 : 252–259, 2015     

Physical exercise during muscle regeneration improves recovery of the slow/oxidative phenotype

Introduction: As skeletal muscle mass recovery after extensive injury is improved by contractile activity, we explored whether concomitant exercise accelerates recovery of the contractile and metabolic phenotypes after muscle injury. Methods: After notexin‐induced degeneration of a soleus muscle, Wistar rats were assigned to active (running exercise) or sedentary groups. Myosin heavy chains (MHC), metabolic enzymes, and calcineurin were studied during muscle regeneration at different time points. Results: The mature MHC profile recovered earlier in active rats (21 days after injury) than in sedentary rats (42 days). Calcineurin was higher in the active degenerated than in the sedentary degenerated muscles at day 14. Citrate synthase and total lactate dehydrogenase (LDH) activity decreased after injury and were similarly recovered in both active and sedentary groups at 14 or 42 days, respectively. H‐LDH isozyme activity recovered earlier in the active rats. Conclusions: Exercise improved recovery of the slow/oxidative phenotype after soleus muscle injury. Muscle Nerve 55 : 91–100, 2017